Genetic engineering of lymphocytes to express a transgenic T ceil receptor (TCR) specific for a defined melanoma antigen allows the efficient generation of large populations of antigen-specific T cells suitable for T cell adoptive immunotherapy. These engineered cells can be additionally genetically labeled with a positron emitting tomography (PET) reporter gene to allow the non-invasive study of the in vivo kinetics of antitumor T cell responses. In this second revised project we propose a clinical trial incorporating advances from our ongoing preclinical studies. We propose to deplete CD25+ cells (including natural T regulatory -nTreg- cells) before activation and to transduce peripheral blood mononuclear cells (PBMC, including both CD8+ and CD4+ T cells) with a high affinity TCR to the melanoma tumor rejection antigen MART-1 using a lentiviral vector. We will follow the bio-distribution and persistence of adoptively transferred, transduced PBMC in vivo via PETbased imaging and relate this to their functional activity, assessed by cutting edge immunoassays. Aim 1 will be a phase I dose escalation clinical trial that will test the safety and feasibility of adoptive transfer of MART-1 TCR transgenic CD8+ and CD4+ T cells to patients with metastatic melanoma. All patients will be HLA-A2.1 positive and have MART-1 expressing melanoma. Two cohorts of 6 patients will have received a lymphodepleting or myelodepleting chemo-irradiation regimen to study how to fully exploit post-conditioning homeostatic T cell proliferation. Following adoptive TCR transgenic cell transfer, patients will receive MART-1- specific in vivo boosting with dendritic cell (DC) vaccines together with high dose interleukin-2 (IL-2) to foster additional antigen-driven expansion. Patients in the myelodepleting cohort will receive hematopoietic support with the reinfusion of CD34-purified peripheral blood stem cells (PBSC). In Aim 2 we will examine the impact of the conditioning regimen and the infused TCR transgenic cell phenotype on the ex vivo expansion, persistence, phenotype and function of MART-1 TCR transgenic CD8+ T cells recovered from patients. We hypothesize that the T cell subpopulations that are able to sustain long-term persistence (naive and central memory T cells) will have adequate systemic distribution, tumor targeting and antitumor activity. To evaluate this premise, we propose to study serially sampled blood and tumors for immune function assessment. PET-based imaging technology will guide us to sample tumor sites infiltrated with the TCR transgenic T cells. In Aim 3 we will investigate the contribution to antitumor immunity of CD4+ cells genetically modified to recognize a tumor antigen restricted by MHC class I molecules (a cell type that does not exist in nature). We will determine their ability to serve as effective helper cells for CD8+ cytotoxic T lymphocytes (CTL) and rule out the possibility that they become induced suppressor Treg (iTreg). In summary, we propose to test tumor antigen-specific TCR transgenic cell adoptive transfer in a bench-tobedside and bedside-to-bench clinical research project.